The
present work evaluated the cytotoxicity of methanolic and aqueous extracts of rhizome of Acoruscalamus Linn.
which is a well known medicinal plant used in traditional treatment. Both
extracts were found to be cytotoxic as determined
by Alliumcepa root
tip assay and XTT assay in MDA-MB-435S and Hep3B cell lines. Results of the
study indicate that the plant possesses anti-tumor properties and may serve
as a potential source for investigation and development of anti-cancer drugs.

������� A wide variety of secondary
metabolites obtained from plants are tested for their ability to treat
cancer. Various anti-cancer drugs from plants are known to be effective
against proliferating cells. They exhibit cytotoxic
effects either by damaging DNA or by blocking the formation of mitotic
spindle during stages of cell division (Gali-Muhtasib
and Bakkar, 2002). However most of the cytotoxic drugs exhibit side effects, and hence, there is
a need for drugs that are efficient and have less side effects (Powis, 1983). The use of plant products in the treatment
of cancer has been of recent interest (Bauer, 2000). Many drugs that are used
for cancer treatment are at present obtained from plant sources. These
include the well known vincaalkaloids, vincristine and vinblastin, isolated from Catharanthusroseus, etoposide and teniposide,
derivatives of epipodophyllotoxin, isolated from Podophyllum,
and several others (Cragget al., 1993, 1994; Wang, 1998).

�������� Vincristine
sulfate was purchased from Sigma-Aldrich. Colchicine,
L-15 (Leibovitz) medium with L-glutamine, Minimal
Essential Medium (MEM) Eagle with Earle�s salt, NEAA and L-glutamine and phenazinemethosulphate (PMS)
were purchased from Hi-Media (Mumbai, India). MDA-MB-435S (human breast
carcinoma) and Hep3B (human liver carcinoma) cell lines were obtained from
National Center for Cell Science (NCCS), Pune,
India.� All other chemicals and
solvents were of the highest commercial grade and used without further
purification.

Collection of plants

�������� Acoruscalamus rhizomes were
obtained from its natural habitat in Coimbatore region, Tamil Nadu, India in
the month of December, 2007. They were shade-dried, powdered, sieved and
stored prior to further use. Voucher specimens were maintained in our
laboratory for future reference.

Extraction

��������� Rhizome powder was extracted sequentially
with methanol and water using a Soxhlet apparatus
and evaporated to dryness in vacuum at 60�C in a rotary evaporator (Buchi, Switzerland) (Saeedehet al., 2007). �The dried extracts in suitable
concentrations were tested for cytotoxicity.

Alliumcepa
root tip assay

�������� Alliumcepa root tip meristems
have been widely used for the testing of cytotoxicity
and anti-mitotic activity of various compounds (Grant et al., 1981). A. cepa root tip assay was introduced by Levan in 1938 and was later proposed as a standard method
to study genotoxicity (Fiskesjo,
1985). 100 g/ml, 200 g/ml,
500 g/ml and 1000 g/ml
of the extracts were used for the assay. Bases of onion bulbs were suspended in
extract solutions. At the end of exposure periods (48 and 72 h), root lengths
were measured (in cms) with a ruler. Onions grown
on double distilled water (without any extract) served as the negative
control. Vincristine and colchicine
were employed as positive controls (data not shown).

Mitotic index calculation

������� Mitotic index was calculated as
described by Bloch et al. (1967). Roots
were excised at 48 and 72 h, and suspended in a fixative (ethanol: acetic
acid = 3:1) for 10 min. They were then washed with distilled water,
hydrolyzed at 60 �C in 1N HCl for 15 min, and
stained with 0.5% hematoxylin stain for 10 min. Stained
root tips were excised and squashed on a clean glass slide with a drop of 45%
acetic acid and examined under microscope. In all the slides, 400-500 cells
were counted to determine the number of cells in interphase
and dividing phase (Sehgalet al., 2006).� Mitotic index was calculated by using the
formula:

Mitotic
index percentage (%MI) =

Total
number of cells in mitosis

X
100

Total
number of cells counted

Analysis of cytotoxicity:
XTT assay

������� XTT assay was performed on MDA-MB-435S
(grown in L-15 medium) and Hep3B (grown in MEM medium) cell lines as
described by Weislowet al. (1989).� 6 X 103
cells were seeded on 96-well plates and the cells were supplemented with
200 l of the respective culture medium for a
period of 24 h. The media were then substituted by 200 l
of fresh media containing varying concentrations of the extracts (15.625 g/ml, 31.25 g/ml,
62.5 g/ml and 125 g/ml).
The plates were incubated at 37 �C for 24 h, after which, media were removed
and fresh media were added. 50 l of XTT
reagent prepared in medium (0.6 mg/ml) containing 25 M
of PMS was then added to all the wells and the plates were incubated in dark humid
conditions at 37 �C for 4 h.� After
incubation, the orange colored complex formed was read at 450 nm using a DynexOpsys MRTMMicroplate Reader (Dynex
Technologies, VA, USA) with a 630 nm reference filter. Wells containing cells
without extract treatments served as the control. Wells containing only
culture medium and XTT reagent served as the blank. Percentage cytotoxicity of the extracts was calculated by using the
formula:

�

%
Cytotoxicity =

(OD
of control � OD of treated cells)

X
100

OD
of control

Statistical analysis

����� ����All data were recorded as mean � standard
deviation of triplicate measurements.� Significant
differences among treatment means were determined by ANOVA at P<0.05.
MATLAB ver. 7.0 (Natick, MA, USA) and Microsoft Excel 2007 (Roselle, IL, USA)
were used for the statistical evaluations.

������������ The
inhibitory effects of the extracts were evaluated on the growth of Alliumcepa root meristem. Root lengths and % mitotic index (in root tip
cells) in control group and treated groups at 48 h and 72 h are given in Table
1.

Table 1: Root
length and % mitotic index of root tip cells of Alliumcepaat 48
h and 72 h of exposure to different concentrations of aqueous and methanolic extracts of A. calamus.

Name
of the extract

48
hrs

72
hrs

Root
length (in cms)a

%
MI

Root
length (in cms)a

%
MI

Control

1.87
� 0.35 (n=29)

72.68

2.82
� 0.92

(n=38)

69.72

Acoruscalamus aqueous extract

100
�g/ml

1.82
�� 0.61

(n=15)

70.42

2.32
�� 0.64

(n=22)

67.59

200
�g/ml

1.55
� 0.91

(n=8)

65.02

1.96
�� 0.50

(n=19)

60.36

500
�g/ml

0.97
� 0.24

(n=18)

50.18

1.09
�� 0.15

(n=22)

58.04

1000
�g/ml

0.62
� 0.29

(n=20)

37.62

0.75
�� 0.16

(n=11)

23.63

Acoruscalamusmethanolic extract

100
�g/ml

1.88
� 0.35

(n=22)

67.92

1.86
� 0.33

(n=25)

66.02

200
�g/ml

1.40
� 0.2

(n=31)

64.02

1.25
� 0.78

(n=20)

60.61

500
�g/ml

0.92
� 0.29

(n=21)

56.12

1.02
� 0.06

(n=15)

58.72

1000
�g/ml

0.75
� 0.28

(n=16)

34.62

0.89
� 0.25

(n=20)

35.49

a
mean root length � SD at 95%
confidence interval

XTT assay

Table 2 presents the IC50 values for the aqueous and methanolic extracts of A. calamus in MDA-MB-435S and Hep3B
cell lines. Although the magnitude of cytotoxicity
varied among the two cell lines, considerable cytotoxicity
was demonstrated in both cell lines, thereby indicating the presence of anti-cancer
metabolites.

Table 2:� IC50 values of aqueous and methanolic extracts of A. calamus in MDA-MB-435S and Hep3B
cell lines as determined in XTT assay.

MDA-MB-435S

Hep3B

Aqueous extract

63.65 � 8.30 g/ml

85.22 � 11.40 g/ml

Methanolic extract

13.71 � 6.66 g/ml

32.74 � 4.55 g/ml

DISCUSSION

�������� In this study, both extracts of A. calamusrhizome
showed cytotoxicity. The results indicate that
these plant products might act against development of cancer. There was a significant
concentration and time dependent decrease (P<0.05) in A. cepa root length along with a
simultaneous decline in percentage mitotic index of root tip cells.

The
extracts demonstrated congruous trend of cytotoxicity
in all test models. Correlations between results from all test systems were
analyzed (P<0.05). Methanolic extracts showed significant positive correlation� between cytotoxicity
demonstrated in MDA-MB-435S and A. ceparoot
tip cells for both 48 h (r2 = 0.99276) and 72 h (r2 =
0.9901) treatments; as well as cytotoxicity between
Hep3B and A. ceparoot tip cells [r2
= 0.982278 (48 h); r2 = 0.976605 (72 h)]. Aqueous extract, on the
other hand, did not demonstrate any significant correlation with respect to cytotoxicity between A.
ceparoot tip cells and either of the cell
lines. Furthermore, cytotoxicity caused in
MDA-MB-435S and in Hep3B cell lines by the methanolic
extract have a significant positive mutual correlation (r2 =
0.986297694), which is lacking in case of the aqueous extract. These
differences observed in correlation might be due to diverse components �with differential activities towards various
cell types being eluted in the solvents employed.

According to the American National Cancer Institute, the IC50
limit to consider a crude extract promising for further purification is
lower than 30 �g/ml (Suffness and Pezzuto, 1990). IC50 values of the methanolic extract for both cell lines are well within
this limit. These results strongly
prove that methanolic extract of A. calamus
has a strong and consistent anti-proliferative effect on plant tissue cells
and animal cell lines.

Conclusion

����������� The study concludes that rhizome
of Acoruscalamus
might be considered as a potential source of metabolites which could be developed
as precursors for anti-cancer drugs. Isolation and purification of these
active compounds are in prospect.

Acknowledgement

We
are thankful to the management of VIT University, Vellore, Tamil Nadu, India,
for providing the necessary facilities and infrastructure for the successful
accomplishment of this work.